IgG-specific Enzyme Immunoassay Research Articles

Heparin Induced Thrombocytopenia: A Laboratory Comparison of the IgG Specific EIA Vs Polyspecific EIA

Introduction:Heparin-induced Thrombocytopenia (HIT) is a potentially life/limb threatening immune mediated pro-thrombotic syndrome triggered by heparin/platelet factor 4 (PF4) complexes. Based on clinical probability, if indicated, initial laboratory testing for HIT is typically based on enzyme immunoassay (EIA) to detect anti-PF4/heparin(H) antibodies with or without supplemental functional assays, the serotonin release assay (SRA) is considered to be the gold standard. EIAs detect either polyspecific (IgG, IgA, IgM) or IgG specific antibodies, the latter are recommended by ISTH/SSC, and should have optimal performance characteristics (sensitivity/specificity/negative and positive predictive values (NPV/PPV)). Each performing laboratory, however, needs to establish in-house performance characteristics of the assay.Objective:To compare laboratory performance characteristics of IgG specific and polyspecific PF4/H EIAs for a positive SRA.Materials and methods: Between 2013 to 2017, 284 serum samples submitted to our laboratory for clinical HIT EIA testing were studied. IgG specific and polyspecific (Immucor GTI Diagnostics, Inc. Waukesha, WI) assays were performed per the manufacturer's instructions with the exception of performance on a liquid handling instrument rather than manually. The manufacturer assigned optical density (OD) cutoff of ≥0.400 was defined as positive. The SRA, ordered separately by clinicians, were sent out to reference laboratories; criteria for a positive result was ≥20% serotonin release with low dose heparin and <20% release with high dose of heparin. The data were entered into Excel spreadsheet, de-identified and analyzed by Analyse-it (Analyse-it Software, LTD, Leeds, U.K.). We also correlated the SRA positivity with the EIA ODs.Results:Of the 284 serum samples, 112 were positive with IgG specific EIA, 68 (60.7%) of those also had a positive SRA. 172 were negative with IgG specific EIA, 2 (1.2%) of those had a positive SRA. Of the 148 were positive with polyspecific EIA, 69 (46.6%) also had a positive SRA. 136 were negative with polyspecific EIA, 1 (0.7%) had a positive SRA. The performance characteristics are shown in table 1. Compared with the polyspecific EIA, the IgG specific EIA had superior specificity, PPV and positive likelihood ratio (LR); the NPVs and negative LR of both assays were similar. The percent of patients having a positive SRA at different OD cutoffs is illustrated in Table 2.Conclusion: In aggregate the data suggest that the IgG specific EIA had superior performance characteristics in detecting HIT antibodies. Given the high likelihood of a positive SRA for OD >2.0, in the appropriate clinical context, supplemental SRA testing may not be essential. Since the data were de-identified, assay performance for the clinical diagnosis of HIT was not possible. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of antibodies in human immunoglobulin products from different regions worldwide

AimThe antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. MethodsTwenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in 10 countries/regions. Depending on each pathogen, antibody levels were measured using specific commercial IgG-specific enzyme immunoassay kits or by cell culture neutralization test and guinea pig skin neutralization test. A principal component analysis was performed. ResultsFor polio and diphtheria (reference markers of the US authorities), all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia, and the USA were positive for titers against globally distributed pathogens or those under vaccination programs in the developed world (parainfluenza, Epstein–Barr, varicella-zoster, influenza B, parvovirus B19, and measles viruses). IVIG from Taiwan and Hong Kong showed low antibody titers for these pathogens but high titers for Pseudomonas aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, and hepatitis E viruses and Streptococcus pneumoniae). IVIGs from Argentina, Spain, Israel, and Czechia showed intermediate antibody concentrations. ConclusionThe antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs, and the prevalence of pathogens in the different countries and regions.

Evaluation of a flow cytometer-based functional assay using platelet-rich plasma in the diagnosis of heparin-induced thrombocytopenia

BackgroundHeparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT. Study designSera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test. Results390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA. ConclusionThe Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls.

Laboratory diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a clinical-pathological disorder; thus, laboratory testing for the pathogenic platelet-activating antiplatelet factor 4 (PF4)/heparin antibodies is central for diagnosis. The "iceberg" model summarizes the inter-relationship between platelet activation assays and PF4-dependent immunoassays, with platelet-activating antibodies comprising a subset of anti-PF4/heparin antibodies. The platelet serotonin-release assay (SRA), performed by reference laboratories, has high sensitivity and specificity for HIT (~95% each), and is especially suited for detecting highly pathogenic HIT sera containing both heparin-dependent and heparin-independent platelet-activating antibodies; this latter subgroup of antibodies explains "autoimmune HIT" disorders (delayed-onset, persisting, spontaneous, heparin "flush," fondaparinux-associated). Recently, SRA-negative HIT has become recognized, in which serum from some HIT patients contains subthreshold levels of platelet-activating antibodies (by SRA) that become detectable using a PF4-enhanced platelet activation assay. Unusual immunologic features of HIT include early antibody detectability (at onset of platelet count fall) and antibody transience (seroreversion). Widely available PF4-dependent enzyme immunoassays (EIAs) have high sensitivity but poor specificity for HIT, although specificity is enhanced with IgG-specific EIAs and strong positive results; unfortunately, EIA results are usually not available in real time. Automated rapid immunoassays, such as the chemiluminescence immunoassay (CLIA) and latex immunoturbidimetric assay (LIA), facilitate real-time laboratory diagnosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR-) test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian analysis, into real-time posttest probabilities that are dramatically increased (test positive) or decreased (test negative). Moreover, (semi-)quantitative CLIA- and LIA-positive results (weak, moderate, strong positive) can further refine the posttest probability of HIT.

Heparin-induced thrombocytopaenia diagnostic testing after adult cardiac surgery: a single-centre experience and development.

To assess the incidence and outcome of heparin-induced thrombocytopaenia (HIT) in patients after cardiac surgery on cardiopulmonary bypass (CPB) and to review the time course of platelet counts and the use of different immunological diagnostic tests. All patients ≥18 years of age who underwent CPB from 2006 to 2015 and who were postoperatively admitted to our cardiac intensive care unit (ICU) were included in this retrospective study. Screening for heparin/platelet factor-4 antibodies was performed using an antibody test, which was later replaced with a screening test specific for IgG antibodies without IgA/IgM cross-reactivity. The enzyme immunoassay (EIA) for the detection of antibodies of all immunoglobulin classes against heparin/PF4 complexes was replaced with an IgG-specific EIA. HIT was confirmed by a heparin-induced platelet aggregation test until 2014. Among 4978 patients admitted between 2006 and 2015, 539 (11%) patients were evaluated for HIT. Patients were excluded because of age <18 years (n = 9), non-cardiac surgery without CPB (n = 10) or incomplete data (n = 3). Of the remaining 517 patients, 43 (8.3%) patients were HIT-positive. HIT incidence was 0.86%. The proportion of HIT-positive patients was similar in men and women (8.4% and 8.2%, respectively). Men and women with suspected HIT also had similar in-hospital mortality (odds ratio ≈ 1; P = 0.926). The incidence of HIT was lower in our study than previously reported. Novel immunological tests have improved to specifically detect IgG antibodies. Furthermore, they are able to detect anti-protamine antibodies, which may be present in patients with high clinical probability of testing negative for HIT. Incidence and clinical relevance of heparin/protamine antibodies will be subjects of future investigation.

Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities

AbstractRepeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera—when first testing SRA-negative—continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive.

Low Level Platelet-Activating Antibodies in Patients Suspected of Having Heparin-Induced Thrombocytopenia but Testing Negative in the ”Gold Standard” Functional Test

Background: Heparin-induced thrombocytopenia (HIT) is a common drug reaction that causes arterial or venous thrombosis as a result of heparin therapy. Platelet-activating antibodies, against complexes of platelet factor 4 (PF4) and heparin, cause intense platelet activation, ultimately leading to an increased risk of thrombosis, limb-loss and even death. Most patients exposed to heparin will produce non-pathogenic anti-PF4/heparin antibodies while only a small number will produce platelet-activating and HIT-causing antibodies (pathogenic HIT antibodies). Among HIT tests, the functional assays, such as the serotonin release assay (SRA), correlate best with the disease because they can specifically identify the pathogenic HIT antibodies whereas the enzyme immunoassays (EIAs) cannot. We have previously shown that anti-PF4/heparin antibody production precedes thrombocytopenia in HIT patients (Warkentin et al., Blood 2009 113: 4963-4969) possibly indicating the need for a threshold plasma level of pathogenic HIT antibody, among other factors, to cause the disease. The objective of this study was to investigate the presence of low levels of pathogenic HIT antibodies in samples from patients suspected of HIT who had detectable anti-PF4/heparin antibodies in the EIA (EIA-positive), but who did not have platelet-activating antibodies in the standard SRA (SRA-negative).Methods: We used an in-house IgG-specific EIA to detect the presence of anti-PF4/heparin antibodies (EIA-positive: OD405nm> 0.45) and the standard SRA to detect the presence of heparin-dependent platelet-activating antibodies (SRA-positive: release >20% with 0.1-0.3 IU/mL of unfractionated heparin). We developed an enhanced SRA (eSRA) by adding increasing concentrations of exogenous PF4 (0-100 μg/mL) to detect sub-threshold levels of platelet activating antibodies undetectable in the standard SRA (eSRA-positive: release >20%). Samples tested were referred for HIT testing by the McMaster Platelet Immunology Laboratory (Hamilton, Canada).Results: Sera from healthy individuals (n=10) and from suspected HIT patients with a negative anti-PF4/heparin EIA (n=15) did not demonstrate platelet activation in the eSRA at any dose of exogenous PF4 added. SRA-positive sera (n = 7), diluted sufficiently that they were non-reactive in the standard SRA, demonstrated PF4 dose-dependent platelet activation in the eSRA. This confirmed the increased sensitivity of the eSRA in detecting low-titre platelet-activating antibodies. Reactivity in the eSRA was inhibited by high heparin (100 U/mL) and by blocking the platelet FcgRIIa receptor with the monoclonal antibody IV.3. We then tested samples (n=24) referred for HIT testing that were positive in the anti-PF4/heparin EIA (optical densities OD405nm 0.7 to 2.4) but negative in the standard SRA. Heparin-dependent platelet activation (20-99% release) was demonstrated in 11 of 24 (46%) in the eSRA. This reactivity directly correlated with the amount of PF4 added to the platelets (optimal concentration of PF4 12.5 - 100 μg/mL) but not with the strength (OD405nm) of the anti-PF4/heparin EIA. In further investigations, we concentrated (4-fold) 7 of the 11 eSRA-positive samples in an attempt to increase the concentration of the antibodies. Of those 7 samples, 5 (71%) became positive in the standard SRA upon testing of the concentrated sample.Conclusions: These data indicate that low-titre platelet-activating antibodies may be found in some patients suspected of having HIT that test negative in the standard SRA irrespective of the strength (OD405nm) of the anti-PF4/heparin EIA. The immune response during heparin therapy can produce both families of pathogenic and non-pathogenic anti-PF4/heparin antibodies but it is the titre of the pathogenic antibody that may be necessary for platelet activation. Perhaps under permissive clinical conditions and with patient-specific factors, the titre of the pathogenic HIT antibodies may increase and lead to HIT. DisclosuresWarkentin:Pfizer Canada: Honoraria; Instrumentation Laboratory: Honoraria; GlaxoSmithKline: Consultancy, Research Funding; W.L. Gore: Consultancy, Research Funding.

Incidence of Direct Thrombin Inhibitor Therapy for Presumed Heparin Induced Thrombocytopenia Before and After Implementation of Heparin Induced Thrombocytopenia IgG Subtype Antibody Test

AbstractAbstract 3336 Introduction:Heparin Induced thrombocytopenia (HIT) is a clinicopathological entity in which thrombocytopenia occurs in temporal relation to the initiation of heparin therapy. The diagnosis is made using clinical criteria and a scoring system (4T) can be applied to assess the probability of the diagnosis. A variety of sensitive tests are available but the most widely applied is an enzyme immunoassay (EIA), which detects and semi-quantifies antibodies of any class to the heparin-PF4 complex. The role of testing is largely supplementary, but recently focus has been placed on the epidemic of HIT overdiagnosis due to overreliance on laboratory tests irrespective of the clinical scenario. A newer EIA can measure the pathogenic IgG antibody only. This study examines the number of patients started on direct thrombin inhibitor (DTI) therapy for a presumptive diagnosis of HIT prior to the implementation of the IgG antibody and after its introduction at one large academic tertiary care center. Methods:All patients placed on the DTIs argatroban and lepirudin between October 2006-May 2009 in the pre IgG subtype antibody period and May 2009-February 2012 in the post IgG subtype period for a presumptive diagnosis of HIT were identified. Demographic data including age, sex, and primary service were recorded. All HIT antibody (Ab) immunoglobulin class EIA and IgG specific EIA optical density were recorded, as available. 4T criteria and rates of thrombosis were assessed retrospectively. Incidence of WHO Grade III (requiring transfusion) or IV (life threatening) bleeding was documented. Chi Square, Mann Whitney U, and Fisher's exact tests were used as appropriate for statistical analysis. Results:89 patients pre availability of the IgG EIA assay and 100 patients post availability of the IgG EIA assay met inclusion criteria and were evaluated. Groups were matched in terms of sex and 4T pretest probability distribution as well as rates of thrombosis. Patients post IgG EIA availability were older and had lower mean total HIT Ab optical densities. Post IgG EIA patients were treated with DTI therapy 5 days less, which was statistically significant. The incidence of Grade III and IV bleeding episodes, however, was statistically similar between groups. In the pre IgG EIA group, 7 bleeds (4 Grade III, 3 Grade IV) occurred in patients with low pretest probability for HIT based on 4T criteria. In the post IgG EIA group, 8 bleeds (5 Grade III, 3 Grade IV) occurred in patients with low pretest probability for HIT based on 4T criteria.Table 1Comparison between demographic and clinical outcome data derived from patients treated with DTI therapy for presumed HIT prior to IgG EIA availability and post IgG EIA availabilityPre IgG EIA (N=89)Post IgG EIA (N=100)P valueMedian Age (Range)60.5 (18–91)67 (25–98)0.03M:F46:4354:460.754T Pretest ProbabilityLow34460.28Intermediate44400.19High11140.74Median Total Antibody Optical Density0.620.490.02Median Days on Direct Thrombin Inhibitors (DTIs)83<0.0001Grade III/IV Episodes of Bleeding20220.92Grade III12170.50Grade IV850.28Episodes of Thrombosis16171 Conclusion:Implementation of the IgG HIT EIA resulted in a decrease in the number of days on a DTI without an observed change in serious bleeding events, indicating that such bleeding occurs early after initiation of DTI therapy. Further, patients pre and post availability of the IgG EIA were started on DTI therapy despite a low pretest clinical risk for HIT. The use of a test with a shorter turn-around time to exclude HIT or more rigorous enforcement of clinical guidelines will be required to minimize misdiagnosis and avoid therapy related morbidity. Disclosures:No relevant conflicts of interest to declare.

Assessment of serological evidence for mumps virus infection in vaccinated children

It is estimated that at least one-third of mumps virus infections in non-vaccinated individuals are asymptomatic. Little information is available whether this proportion is the same among those vaccinated. We validated a commercial oral fluid mumps IgG-specific Enzyme Immunoassays (EIA) with vaccinated control groups to identify symptomatic and asymptomatic mumps virus infections in vaccinated individuals during a mumps outbreak in The Netherlands. A vaccinated control group was required to define a new cutoff value for the assay, because of the presence of low but significant levels of IgG antibodies in oral fluid as a result of mumps vaccination in the past. With a new cutoff, calculated using receiver operator characteristic analysis, we identified an attack rate of 7-10% compared to 2.7% based on clinical symptoms among vaccinated children. This finding has important implications when studying transmission patterns, strain virulence, as well as mumps vaccine effectiveness to protect from infection rather than disease.

Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay

Background IgG-specific anti-PF4/heparin enzyme-immunoassays (EIAs) are sensitive but not specific for platelet-activating antibodies, the cause of heparin-induced thrombocytopenia (HIT). Two features of EIA reactivity predict for presence of HIT antibodies - the magnitude of a positive result (in optical density [OD] units) and the inhibition of reactivity at high heparin concentrations - but their combined utility remains uncertain. Objective To determine for an IgG-specific EIA how the OD values of a positive reaction and its inhibition by high heparin can be optimally combined. Methods We screened 1,000 consecutive patients with suspected HIT using an IgG-specific PF4/heparin in-house EIA with and without high heparin (100 IU/mL); and by the heparin-induced platelet activation test. Results Platelet-activating antibodies were rarely detected (< 0.2%) when the IgG-specific EIA was negative at the conventional cut-off (OD, 0.5). However, an OD cut-off of 1.0 resulted in an unacceptable loss of sensitivity (14/83 = 17%) for detecting platelet-activating antibodies. The high heparin step increased specificity for platelet-activating antibodies from 72% to 89% without loss of sensitivity when applied to weak-positive sera (OD ≤ 1.0). However, decreased sensitivity was observed with strong-positive sera (OD > 1.0): 11/69 such sera (16%) that did not show > 40% inhibition by high heparin nevertheless contained platelet-activating antibodies. Conclusion Specificity of an IgG-specific EIA for detecting platelet-activating antibodies can be optimized by applying the high heparin inhibition step to weak-positive reactions (0.5- ≤ 1.0 OD). However, applying the high heparin inhibition step to strong-positive reactions (> 1.0 OD) in our in-house assay risks falsely classifying a serum as negative for platelet-activating antibodies.

Presence and utility of IgA-class antibodies to cyclic citrullinated peptides in early rheumatoid arthritis: the Swedish TIRA project

IntroductionThe present study was carried out to assess whether IgA-class antibodies against cyclic citrullinated peptides (IgA anti-CCP) in recent-onset rheumatoid arthritis add diagnostic and/or prognostic information to IgG anti-CCP analysis.MethodsSerum samples were obtained from 228 patients with recent-onset (<12 months) rheumatoid arthritis at the time of inclusion in the Swedish TIRA cohort (Swedish Early Intervention in Rheumatoid Arthritis). Sera from 72 of these patients were also available at the 3-year follow-up. Disease activity and functional ability measures (erythrocyte sedimentation rate, serum C-reactive protein, 28-joint count Disease Activity Score, physician's assessment of disease activity, and the Swedish version of the Health Assessment Questionnaire) were registered at inclusion and at regular follow-ups during 3 years. An IgA anti-CCP assay was developed based on the commercially available IgG-specific enzyme immunoassay from EuroDiagnostica (Arnhem, the Netherlands), replacing the detection antibody by an anti-human-IgA antibody. A positive IgA anti-CCP test was defined by the 99th percentile among healthy blood donors.ResultsAt baseline, a positive IgA anti-CCP test was observed in 29% of the patient sera, all of which also tested positive for IgG anti-CCP at a higher average level than sera containing IgG anti-CCP alone. The IgA anti-CCP-positive patients had significantly higher disease activity over time compared with the IgA anti-CCP-negative patients. After considering the IgG anti-CCP level, the disease activity also tended to be higher in the IgA anti-CCP-positive cases – although this difference did not reach statistical significance. The proportion of IgA anti-CCP-positive patients was significantly larger among smokers than among nonsmokers.ConclusionAnti-CCP antibodies of the IgA class were found in about one-third of patients with recent-onset rheumatoid arthritis, all of whom also had IgG anti-CCP. The occurrence of IgA-class antibodies was associated with smoking, and IgA anti-CCP-positive patients had a more severe disease course over 3 years compared with IgA anti-CCP-negative cases. Although IgA anti-CCP analysis does not seem to offer any diagnostic information in addition to IgG anti-CCP analysis, further efforts are justified to investigate the prognostic implications.

Intrafamilial transmission of Helicobacter pylori: the association between a parent and an offspring with respect to the presence of anti-CagA antibody.

In this study, we examined whether the transmission of Helicobacter pylori occurred in married couples. We also examined whether a correlation existed between H. pylori seropositivity in a parent and an offspring, which may be related to H. pylori transmission. A cross-sectional study of residents living in a rural area in Japan was conducted in 1998. The study population consisted of 1910 volunteers, aged from 22 to 79 years, residing in this area. We serologically confirmed the presence of the antigen, CagA (cytotoxin-associated gene A), of H. pylori, using a recombinant CagA antigen, in addition to examining for antibodies to H. pylori, as determined by an IgG-specific enzyme immunoassay. The data were analyzed using logistic regression models. A significant association of H. pylori seropositivity was observed ( P < 0.001) in married couples, but no significant difference with respect to the presence of anti-CagA antibodies was observed in the married couples with H. pylori seropositive results ( P = 0.053). The odds ratio was 8.08 (95% confidence interval [CI], 1.39-40.05) for infection in offspring with seropositive mothers and 2.93 (95% CI, 0.25-33.85) for infection in offspring with seropositive fathers when compared with seronegative fathers or mothers as the reference. There was a concordance between the presence or absence of anti-CagA antibodies in 11 of 13 groups of mothers and offspring (84.6%) compared with concordance in 6 of 14 groups of fathers and offspring (42.9%; P < 0.05). Our results suggest that maternal influence is likely to be more powerful than paternal influence in the transmission of H. pylori.

Study of booster doses of two Haemophilus influenzae type b conjugate vaccines including their interchangeability

A prospective, two center study of 319 children was undertaken to assess responses to booster immunization in healthy 18-month-olds who completed primary immunization 12 months earlier with Haemophilus influenzae type b (Hib) conjugate vaccine (either HbOC or PRP-T). Interchangeability of these products as boosters was also assessed, using combination products containing diphtheria, pertussis and tetanus components. The study was randomized and evaluator blinded. Sera obtained prior to booster vaccination and 4 weeks later were tested for anti-PRP using an IgG-specific enzyme immunoassay. Pre-immunization anti-PRP levels were low: unmeasurable levels (<0.06 μg ml −1) were present in 47% of those primed with HbOC and 35% of those primed with PRP-T ( P<0.05). Most children responded strongly to booster vaccination. Interchanging the products had no detrimental effect. Children who lacked measurable antibody prior to re-vaccination had significantly weaker responses 4 weeks afterward. Earlier Hib booster vaccination should be considered in Canada to avoid low anti-PRP trough levels. Extended follow-up of antibody levels and vaccine protective efficacy is also advisable given the observed heterogeneity of responses to booster vaccination.

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection

The fully automated IMx ® immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.

The detection of IgM and IgG antibodies against Babesia bigemina in bovine sera using semi-defined antigens in enzyme immunoassays

Soluble extracts prepared from Babesia bigenina merozoites were tested for anti-genicity in class-specific enzyme immunoassays currently being evaluated for the differential serodiagnosis of bovine babesiosis. Intact merozoites were harvested from erythrocytes from an experimentally-infected calf by controlled hypotonic lysis and differential ultracentrifugation. The merozoites were disrupted by ultrasonication and a crude soluble extract obtained by ultracentrifugation. Fractionation of the crude extract on calibrated Sephadex G-200 column consistently produced 4 fractions with molecular weights of 600, 40, 15 and 5 k (k = 10 3 daltoons). Only the 100 and 15 k fractions proved to be antigenic when reacted against bovine immune sera. These fractions were incorporated into IgM- and IgG-specific enzyme immunoassays and used to determine the kinetics of the host-antibody responses to infection. The use of semi-defined antigens allowed assay standardization and good reproducibility of the results. A calf infected with a cryopreserved stabilate of B. bigemina originating from adult Boophilus microplus ticks developed a mild transient fever from 6–4 days post-infection (d.p.i.) and low parasitaemia levels from 7–16 d.p.i. IgG-antibodies first appeared at 7 d.p.i.,peaked in intensity at 12 d.p.i. and then persisted at these levels until the end of the test period at 49 d.p.i. IgM-antibodies appeared at 7 d.p.i., peaked in intensity from 12–22 d.p.i., but then declined to low levels by 28 d.p.i.. The importance of this transitory IgM-antibody response in the serodiagnosis of acute B. bigemina infections remains to be determined in clinical and field situations.